APPLICANT'S ABSTRACT: The kappa3 opioid receptor is one of several opioid receptors mediating analgesia through a supraspinal mechanism. It can be distinguished from mu, delta, and kappa receptors by antagonist and cross-tolerance studies. The kappa3 receptor agonist naloxone benzoylhydrazone (NalBzoH) is antagonized by nonselective opioid antagonists (Win44,441, diprenorphine, Mr2266), but not by subtype selective antagonists (naloxone, naltrindole, norbinaltorphimine). Tolerance develops to kappa3-mediated analgesia, as it does to all opioids, but there is no evidence of cross-tolerance between kappa3 agonists and opioids selective for other opioid receptor subtypes. Kappa3 receptors have been identified on two human neuroblastoma cell lines, SH-SYSY and BE(2)-C, and inhibit forskolin-stimulated cAMP accumulation through pertussis toxin sensitive G-proteins. NalBzoH inhibits cAMP accumulation in SH-SYSY cells at a 1000-fold lower concentration than that required for the same effect in BE(2)-C cells, but its binding affinities for kappa3 receptors in each line are the same. NalBzoH and mu and delta opioids synergistically increase carbachol-stimulated phoshoinositide formation in BE(2)-C cells at a dose (1 nM) that is ineffective at cyclase inhibition, and this effect of NalBzoH is insensitive to toxin treatment. These two events (cyclase inhibition and phosphoinositide formation) may be important biochemical steps involved in kappa3 receptor-mediated analgesia, and are probably regulated by two distinct G-protein alpha-subunits. This application proposes to study the functional regulation of the kappa3 opioid receptor and its intracellular signalling cascades in both neuroblastoma cell lines in comparison with mu and delta receptors in the same cells. These studies will use biochemical, pharmacological, and molecular techniques to monitor receptor affinity and levels, efficacy of cAMP accumulation inhibition and GTPase activity, stimulation of phosphoinositide hydrolysis and cellular cross-talk. My hypothesis is that a specific G-protein subunit present in SH-SY5Y cells is responsible for the 1000-fold difference in kappa3 agonist potency for inhibition of forskolin stimulated cAMP accumulation in the two cell lines. Recent studies indicate that tolerance to mu and kappa agonists is modulated by components of the NMDA receptor signalling pathway, but none of these components affect tolerance to kappa3 agonists. A better understanding of the mechanisms of kappa3 receptor coupling may provide valuable insights into the development of tolerance to kappa3 analgesia, ultimately resulting in more effective treatment for chronic pain.